Mdl-1 ligand

ABSTRACT

The invention provides methods for modulation interactions between MDL-1 and its binding partner, Gal9. Also provided are methods to screen for modulators of MDL-1/Gal9 interaction.

FIELD OF THE INVENTION

The present invention provides the binding partner of MDL-1, MDL-1ligand compositions of matter and uses

BACKGROUND OF THE INVENTION

The control of unwanted immune responses is a critical issue in thetreatment of diseases such as inflammation, autoimmune diseases,transplant rejection, allergic diseases, and some cancers. The activityof overly aggressive T cells can be controlled by immunosuppression orby the induction of immunological tolerance. Tolerance is defined as astate where the immune system is made unresponsive to an antigen,whereas immunosuppression, which decreases the immune response toantigens, usually requires the continued use of medication. Ininflammation and autoimmune diseases, T cells play an essential role inthe prolonged immune response to a certain stimulus. Currentimmunosuppressive regimes commonly involve the use of corticosteroid,cyclosporin or rapamycin, which block the transcription of IL-2, a keygrowth factor for T cells, or inhibit IL-2 dependent proliferation.However, a number of monoclonal antibodies that act as T cell-depletingagents (e.g. CD3, CD4, CD8), or as inhibitors of the cytokine signalingor the co-stimulatory pathways of T cells (e.g. CD25, B7-1, B7-2, CD152,CTLA4) have demonstrated effectiveness in reducing the incidence ofrejection with limited side effects or toxicity. Some of these agentshave been shown to have some degree of success in treating inflammatoryand autoimmune diseases and in prolonging graft survival.

The myeloid receptor of the C-type lectin superfamily associated withDAP12 is Myeloid DAP12-associating Lectin-1 (MDL-1), a type IItransmembrane protein (MDL-1 is also referred to as CLEC5a). MDL-1 wasthe first DAP12 associating molecule to be identified and cloned (Bakkeret al. (1999) PNAS USA 96(17):9792-9796). It is expressed exclusively inmyeloid cells (Bakker et al. (1999) PNAS U.S.A. 96:9792-9796) as well ason other myeloid cell types such as, neutrophils and dendritic cells.The presence of a negatively charged residue in the transmembrane domainof DAP 12 precludes its cell surface expression in the absence of apartner receptor, such as MDL-1, which has a positively charged residuein its transmembrane domain. However, DAP 12 alone is not sufficient forits expression and function at the cell surface. Thus, the combinationof a DAP12-associating molecule, such as MDL-1, and DAP12 may accountfor transmitting a particular physiological signal via DAP12 (Nochi etal. (2003) Am. J. of Pathology 162:1191-1201).

MDL-1 has been found to possibly be the receptor for Dengue Virus onmyeloid cells (see, e.g., Chen, et al. (2008) Nature 453:672-676).Recently, MDL-1 has been structurally characterized as a “C-typelectin-like” homodimeric molecule that is capable of conformationalswitching in the presence of Dengue Virus binding (see, e.g., Watson, etal. (2011) J. Biol. Chem. 286:24208-24218).

The present invention identifies a population of T lymphocyte cells thatappear to express a protein involved in MDL-1 engagement and activation.The ligand appears to be a cell surface protein that may not directlyinteract with MDL-1, but rather involves a third protein. Asub-population of these cells also expresses IL-23 receptor (IL-23R).Upon activation by IL-23, these IL-23R⁺, MDL-1L⁺ expressing cells havebeen implicated in the progression of inflammation, in particularenthesopathy.

The non-viral binding partner of MDL-1, MDL-1 ligand (“MDL-1L”), is nowidentified as Galectin9 (“Gal9”). Galectin-9 (Gal-9) is a member ofanimal lectins that have an affinity to β-galactosides. Gal9 has beenshown to bind to several other molecules, including T cellimmunoglobulin and mucin domain-containing molecule (“TIM3”), which isexpressed on Th1/Th17 cells, and is a negative regulator of Th1 immunity(see, e.g., Zhu, et al. (2005) Nat. Immunol. 6:1245-1252; and Jayaramanet al. (2010) J. Exp Med. 207:2343-2354). Gal9 has also been shown tobind to other cell surface molecules such as CD44 and IgE (Niki, et al.(2009) J. Biol. Chem. 284:32344-32352), as well as protein disulfideisomerase (see, e.g., Bi, et al. (2011) Proc. Natl. Acad. Sci.108:10650-10655).

Engagement MDL-1 by the Gal9 results in the activation of myeloidlineage cells (e.g., macrophages, osteoclasts) via DAP12, resulting intyrosine phosphorylation of DAP12 and induction of an innate immunepathway. Uncontrolled induction of this pathway can either lead tochronic inflammation or improper clearance of infectious microbes. Thusa need exists to control the Gal9/MDL-1 interaction. The presentinvention provides methods to regulate this protein-protein interactionand screens to isolate additional regulatory compositions which modulatewith this interaction.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows that MDL-1-Ig can inhibit IL-23 induced enthesopathy. DX192is an MDL-1 agonist antibody.

FIG. 2 shows that MDL-1-Ig tetramer staining of lymphocytes.

FIG. 3 shows MDL-1-Ig staining of CD45⁺, CD90⁺, and CD117⁺ lymphocytes.

FIG. 4 shows Gal9 treatment exacerbates disease in a murineantibody-induced arthritis model.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery thatGalectin-9 interacts with MDL-1, induces tyrosine phosphorylation ofDAP-12 and stimulates MDL-1 activity on myeloid lineage cells. Further,the present invention identifies a selected population of T lymphocytesthat express a membrane bound protein that binds to Gal9 and throughGal9, engages and modulates MDL-1 activity.

The present invention provides a method of modulating an interactionbetween a lectin-like molecule expressed on a myeloid cell and a lectinwhich binds to a receptor expressed by a T cell, comprising: a)providing a compound that is capable of binding to the lectin-likemolecule at a binding site of the lectin; and b) presenting the compoundof step (i) to the lectin-like molecule and the lectin and therebymodulating the interaction between the lectin-like molecule and thelectin. In certain embodiments, the lectin-like molecule is MDL-1 andthe lectin is Gal9 In a further embodiment, the compound is an antibodyor a binding fragment of an antibody, or a soluble receptor-Ig fusionprotein that modulates the interaction between the lectin-like moleculeand the lectin. The antibody binds MDL-1 and prevents the interaction ofMDL-1 with Gal9.

The present invention also provides a method of screening for a compoundthat modulates an interaction between a lectin-like molecule expressedon myeloid cells, and a lectin which binds to a receptor expressed by aT cell, comprising: a) providing a compound that is capable of bindingto the lectin-like molecule at a binding site of the lectin; and b)presenting the compound of step (a) to the lectin-like molecule and thelectin and thereby modulating the interaction between the lectin-likemolecule and the lectin. In certain embodiments, the lectin-likemolecule is MDL-1 and the lectin is Gal9. In another embodiment, thecompound is an antibody or a binding fragment of an antibody. Theantibody or binding fragment of an antibody inhibits the interaction ofthe lectin-like molecule with the lectin. In a further embodiment, theantibody or binding fragment of the antibody inhibits phosphorylation ofa signaling molecule associated with the lectin-like molecule. In afurther embodiment, the signaling molecule is DAP12 or Syk and thephosphorylation is tyrosine phosphorylation.

The present invention provides A method of depleting a population of Tlymphocyte cells comprising contacting the population of T lymphocytecells with an MDL-1 fusion protein that binds directly or indirectly toa molecule expressed on the T lymphocyte cells. In one embodiment, theMDL-1 fusion protein comprises an extracellular domain of MDL-1 and aheterologous protein. The heterologous protein can be an Fc portion ofan immunoglobulin molecule or human serum albumin. In certainembodiments, the population of T lymphocyte cells express CD45, CD90,and CD117. Additionally the population of T lymphocyte cells can expressIL-23R, which mediates enthesopathy.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

All references cited herein are incorporated by reference to the sameextent as if each individual publication, patent application, or patent,was specifically and individually indicated to be incorporated byreference.

Definitions.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity, to theability to stimulate gene expression, to antigenic activity, to themodulation of activities of other molecules, and the like. “Activity” ofa molecule may also refer to activity in modulating or maintainingcell-to-cell interactions, e.g., adhesion, or activity in maintaining astructure of a cell, e.g., cell membranes or cytoskeleton. “Activity”may also mean specific activity, e.g., [catalytic activity]/[mgprotein], or [immunological activity]/[mg protein], or the like.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibodies of the inventionare described in further detail in U.S. Pat. Nos. 6,090,382; 6,258,562;and 6,509,015, and in U.S. patent application Ser. Nos. 09/801,185 and10/302,356, each of which is incorporated herein by reference in itsentirety.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121 -1123). Theantibody portions of the invention are described in further detail inU.S. Pat. Nos. 6,090,382, 6,258,562, 6,509,015, and in U.S. patentapplication Ser. Nos. 09/801,185 and 10/302,356, each of which isincorporated herein by reference in its entirety.

Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab′, F(ab′)₂, Fabc, Fv, single chains, andsingle-chain antibodies. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

A “conservative amino acid substitution”, as used herein, is one inwhich one amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

As used herein, the term “anti-idiotypic antibodies” or “anti-idiotypes”refers to antibodies directed against the antigen-combining region orvariable region (called the idiotype) of another antibody molecule. Asdisclosed by Jerne et al. (Jerne, N. K., (1974) Ann. Immunol. (Paris)125c:373 and Jerne, N. K., et al., (1982) EMBO 1:234), immunization withan antibody molecule expressing a paratope (antigen-combining site) fora given antigen (e.g., an MDL-1 peptide) will produce a group ofanti-antibodies, some of which share, with the antigen, a complementarystructure to the paratope. Immunization with a subpopulation of theanti-idiotypic antibodies will, in turn, produce a subpopulation ofantibodies or immune cell subsets that are reactive to the initialantigen.

As used herein, the term “fully human antibody” refers to an antibodywhich comprises human immunoglobulin protein sequences only. A fullyhuman antibody may contain murine carbohydrate chains if produced in amouse, in a mouse cell or in a hybridoma derived from a mouse cell.Similarly, “mouse antibody” refers to an antibody which comprises mouseimmunoglobulin sequences only.

“Humanized” antibodies are also within the scope of the presentinvention. As used herein, the term “humanized” or “fully humanized”refers to an antibody that contains the amino acid sequences from thesix complementarity-determining regions (CDRs) of the parent antibody,e.g., a mouse antibody, grafted to a human antibody framework. Humanizedforms of non-human (e.g., murine or chicken) antibodies are chimericimmunoglobulins, which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region of the recipient are replaced byresidues from a complementary determining region of a non-human species(donor antibody), such as mouse, chicken, rat or rabbit, having adesired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are also replaced bycorresponding non-human residues.

As used herein, the term “partially humanized” or “chimeric” antibodymeans an antibody that contains heavy and light chain variable regionsof, e.g., murine origin, joined onto human heavy and light chainconstant regions.

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries contained in transgenicmice, see, e.g., Vaughan et al. (1996) Nat. Biotechnol. 14:309-314;Barbas (1995) Nature Med. 1:837-839; de Haard et al. (1999) J. Biol.Chem. 274:18218-18230; McCafferty et al. (1990) Nature 348:552-554;Clackson et al. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol.Biol. 222:581-597; Mendez et al. (1997) Nature Genet. 15:146-156;Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al.(2001) Phage Display: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Kay et al. (1996) Phage Display ofPeptides and Proteins: A Laboratory Manual, Academic Press, San Diego,Calif.; de Bruin et al. (1999) Nat. Biotechnol. 17:397-399.

As used herein, the term “human” refers to antibodies containing aminoacid sequences that are of 100% human origin, where the antibodies maybe expressed, e.g., in a human, animal, insect, fungal, plant,bacterial, or viral host (Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Clark (2000) Immunol. Today 21:397-402).

The present invention includes “chimeric antibody” which means anantibody that comprises a variable region of the present invention fusedor chimerized with an antibody region (e.g., constant region) fromanother, non-human species (e.g., mouse, horse, rabbit, dog, cow,chicken). These antibodies may be used to modulate the expression oractivity of MDL-1 in the non-human species.

As used herein, the term “human/mouse chimeric antibody” refers to anantibody which comprises a mouse variable region (V_(H) and V_(L)) fusedto a human constant region.

As used herein, the term “single-chain Fv” or “sFv” antibody fragmentsmeans antibody fragment that have the V_(H) and V_(L) domains of anantibody, wherein these domains are present in a single polypeptidechain. Generally, the sFv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains which enables the sFv to formthe desired structure for antigen binding. Techniques described for theproduction of single chain antibodies (U.S. Pat. Nos. 5,476,786,5,132,405 and 4,946,778) may be adapted to produce anti-MDL-1L-specificsingle chain antibodies. For a review of sFv see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, N.Y., pp. 269-315 (1994).

Single chain antibodies, single domain antibodies, and bispecificantibodies are described, see, e.g., Malecki et al. (2002) Proc. Natl.Acad. Sci. USA 99:213-218; Conrath et al. (2001) J. Biol. Chem.276:7346-7350; Desmyter et al. (2001) J. Biol. Chem. 276:26285-26290,Kostelney et al. (1992) J. Immunol. 148:1547-1553; U.S. Pat. Nos.5,932,448; 5,532,210; 6,129,914; 6,133,426; 4,946,778.

As used herein, the terms “disulfide stabilized Fv fragments” and “dsFv”refer to antibody molecules comprising a variable heavy chain (V_(H))and a variable light chain (V_(L)) which are linked by a disulfidebridge.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. AcidsRes. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds MDL-1 is substantially free of antibodies that specifically bindantigens other than MDL-1). An isolated antibody that specifically bindsMDL-1 may, however, have cross-reactivity to other antigens, such asMDL-1 molecules from other species (discussed in further detail below).Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The term “multivalent antibody” refers to an antibody comprising morethan one antigen recognition site. For example, a “bivalent” antibodyhas two antigen recognition sites, whereas a “tetravalent” antibody hasfour antigen recognition sites. The terms “monospecific”, “bispecific”,“trispecific”, “tetraspecific”, etc. refer to the number of differentantigen recognition site specificities (as opposed to the number ofantigen recognition sites) present in a multivalent antibody. Forexample, a “monospecific” antibody's antigen recognition sites all bindthe same epitope. A “bispecific” or “dual specific” antibody has atleast one antigen recognition site that binds a first epitope and atleast one antigen recognition site that binds a second epitope that isdifferent from the first epitope. A “multivalent monospecific” antibodyhas multiple antigen recognition sites that all bind the same epitope. A“multivalent bispecific” antibody has multiple antigen recognitionsites, some number of which bind a first epitope and some number ofwhich bind a second epitope that is different from the first epitope

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFα activity”), is intended to refer to an antibody whosebinding to MDL-1 results in inhibition of the biological activity ofMDL-1. This inhibition of the biological activity of MDL-1 can beassessed by measuring one or more indicators of MDL-1 biologicalactivity, such as MDL-1 induced cytotoxicity (either in vitro or invivo), MDL-1-induced cellular activation and MDL-1 binding to Gal9.These indicators of MDL-1 biological activity can be assessed by one ormore of several standard in vitro or in vivo assays known in the art.

The term “antigen-binding portion” or “antigen-binding fragment” of anantibody (or simply “antibody portion”), as used herein, refers to oneor more fragments of an antibody that retain the ability to specificallybind to an antigen (e.g., hTNFα). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Binding fragments include Fab, Fab′, F(ab′)₂,Fabc, Fv, single chains, and single-chain antibodies. Examples ofbinding fragments encompassed within the term “antigen-binding portion”of an antibody include (i) a Fab fragment, a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the VH andCH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature341:544-546), which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger et al. (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BlAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Example 1 and Joensson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Joensson, U., et al. (1991) Biotechniques11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; andJohnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The term “IC₅₀” as used herein, is intended to refer to theconcentration of the inhibitor required to inhibit the biologicalendpoint.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind MDL-1 is intended to refer to a nucleic acid molecule inwhich the nucleotide sequences encoding the antibody or antibody portionare free of other nucleotide sequences encoding antibodies or antibodyportions that bind antigens other than MDL-1, which other sequences maynaturally flank the nucleic acid in human genomic DNA. Thus, forexample, an isolated nucleic acid of the invention encoding a VH regionof an anti- MDL-1Lantibody contains no other sequences encoding other VHregions that bind antigens other than MDL-1.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “kit” as used herein refers to a packaged product comprisingcomponents with which to administer the anti-MDL-1 antibodies of theinvention for treatment of a MDL-1 related disorder. The kit preferablycomprises a box or container that holds the components of the kit. Thebox or container is affixed with a label or a Food and DrugAdministration approved protocol. The box or container holds componentsof the invention which are preferably contained within plastic,polyethylene, polypropylene, ethylene, or propylene vessels. The vesselscan be capped-tubes or bottles. The kit can also include instructionsfor administering the anti-MDL-1 antibodies or MDL-1 soluble fusionprotein of the invention, e.g., an Ig-MDL-1 fusion.

MDL-1

The invention is directed to methods of modulating myeloid cell activityby modulating the function of the MDL-1, also known as C-type lectinsuperfamily member 5 (CLECSF5), molecules residing on the surface of amyeloid cell, in particular a macrophage, including osteoblasts andosteoclasts. Engagement of MDL-1 with Gal9 herein can also cause theactivation of macrophage cells and the induction of inflammation. Theability to modulate the MDL-1/Gal9 interaction will allow control ofmyeloid inflammation.

The terms “MDL-1”, “Myeloid DAP12 associating lectin-1”, “MyeloidDAP12-associated lectin-1”, DAP-12”, “DAP12”, “DNAX Activation Protein,12 kD” are well known in the art. The human and mouse DAP12 and MDL-1nucleotide and polypeptide sequences are disclosed in WO 99/06557.GenBank® deposits of the human MDL-1 nucleic acid sequence (AR217548)and mouse MDL-1 nucleic and amino acid sequences (AR217549 and AAN21593,respectively) are also available.

The terms “Galectin9”, “Gal9”, “T cell Immunoglobulin Mucin”, and “Tim3”are well known in the art. Polypeptide sequences of human Gal9 areprovided in GenBank® deposits NP_(—)002299 (short form) and NP_(—)033665(long form).

A structural feature of the MDL-1 protein is the extracellular domain,which is defined by amino acid residues 26 to 188 of the human MDL-1protein, and amino acid residues 26 to 190 of the mouse MDL-1 protein.Soluble MDL-1 protein can be fused to heterologous proteins, e.g., theFc portion of an immunoglobulin molecule, or conjugated to chemicalmoieties, e.g., PEG, human serum albumin.

Soluble MLD-1 proteins alone or in combination with heterologousproteins can be used to deplete a population of T lymphocytes. These Tlymphocytes can express certain cell surface molecules including CD45,CD90, and CD117. Additionally, IL-23R can also be expressed by the Tlymphocyte population. Expression of IL-23R allows these cells tomediate enthesopathy.

Molecular Biology

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook, et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

The sequence of any nucleic acid (e.g., a nucleic acid encoding anantibody that modulates the MDL-1/Gal9 interaction) may be sequenced byany method known in the art (e.g., chemical sequencing or enzymaticsequencing). “Chemical sequencing” of DNA may denote methods such asthat of Maxam and Gilbert (1977) (Proc. Natl. Acad. Sci. USA 74:560), inwhich DNA is randomly cleaved using individual base-specific reactions.“Enzymatic sequencing” of DNA may denote methods such as that of Sanger(Sanger et al., (1977) Proc. Natl. Acad. Sci. USA 74:5463).

The nucleic acids herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′- non-coding regions, and the like.

Promoters, which may be used to control gene expression, include, butare not limited to, the cytomegalovirus (CMV) promoter (U.S. Pat.Nos.5,385,839 and 5,168,062), the SV40 early promoter region (Benoist etal., (1981) Nature 290:304-310), the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto et al., (1980) Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., (1981)Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., (1982) Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Komaroff et al., (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731),or the tac promoter (DeBoer et al., (1983) Proc. Natl. Acad. Sci. USA80:21-25); see also “Useful proteins from recombinant bacteria” inScientific American (1980) 242:74-94; and promoter elements from yeastor other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or thealkaline phosphatase promoter.

A coding sequence is “under the control of”, “functionally associatedwith” or “operably associated with” transcriptional and translationalcontrol sequences in a cell when the sequences direct RNA polymerasemediated transcription of the coding sequence into RNA, preferably mRNA,which then may be trans-RNA spliced (if it contains introns) and,optionally, translated into a protein encoded by the coding sequence.

The present invention contemplates modifications, especially anysuperficial or slight modification, to the amino acid or nucleotidesequences that correspond to the proteins. In particular, the presentinvention contemplates sequence conservative variants of the nucleicacids that encode the human MDL-1 and mouse MDL-1 of the invention.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule may anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. Typical low stringency hybridization conditions may be55° C., 5× SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30%formamide, 5× SSC, 0.5% SDS. Typical, moderate stringency hybridizationconditions are similar to the low stringency conditions except thehybridization is carried out in 40% formamide, with 5× or 6× SSC. Highstringency hybridization conditions are similar to low stringencyconditions except the hybridization conditions are carried out in 50%formamide, 5× or 6× SSC and, optionally, at a higher temperature (e.g.,57° C., 59° C., 60° C., 62° C., 63° C., 65° C. or 68° C.). In general,SSC is 0.15M NaCl and 0.015M Na-citrate. Hybridization requires that thetwo nucleic acids contain complementary sequences, although, dependingon the stringency of the hybridization, mismatches between bases arepossible. The appropriate stringency for hybridizing nucleic acidsdepends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the higherthe stringency under which the nucleic acids may hybridize. For hybridsof greater than 100 nucleotides in length, equations for calculating themelting temperature have been derived (see Sambrook et al., supra,9.50-9.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook, et al., supra, 11.7-11.8).

Also included in the present invention are nucleic acids comprisingnucleotide sequences and polypeptides comprising amino acid sequencesthat are at least 70% identical, at least 80% identical, at least 90%identical e.g., 91%, 92%, 93%,94%, and at least 95% identical e.g., 95%,96%, 97%, 98%, 99%, 100%, to the reference nucleotide and amino acidsequences of Table 1 when the comparison is performed by a BLASTalgorithm wherein the parameters of the algorithm are selected to givethe largest match between the respective sequences over the entirelength of the respective reference sequences. Polypeptides comprisingamino acid sequences which are at least 70% similar, at least 80%similar, at least 90% similar e.g., 91%, 92%, 93%, 94%, and at least 95%similar e.g., 95%, 96%, 97%, 98%, 99%, 100%, to the reference amino acidsequences of Table 1 e.g., SEQ ID NOs: 2 and 4, when the comparison isperformed with a BLAST algorithm wherein the parameters of the algorithmare selected to give the largest match between the respective sequencesover the entire length of the respective reference sequences, are alsoincluded in the present invention.

Sequence identity refers to exact matches between the nucleotides oramino acids of two sequences which are being compared. Sequencesimilarity refers to both exact matches between the amino acids of twopolypeptides which are being compared in addition to matches betweennonidentical, biochemically related amino acids. Biochemically relatedamino acids which share similar properties and may be interchangeableare discussed above.

The following references regarding the BLAST algorithm are hereinincorporated by reference: BLAST ALGORITHMS: Altschul et al., (1990) J.Mol. Biol. 215:403-410; Gish et al., (1993) Nature Genet. 3:266-272;Madden et al., (1996) Meth. Enzymol. 266:131-141; Altschul et al.,(1997) Nucleic Acids Res. 25:3389-3402; Zhang et al., (1997) Genome Res.7:649-656; Wootton et al., (1993) Comput. Chem. 17:149-163; Hancock etal., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS:Dayhoff et al., “A model of evolutionary change in proteins.” in Atlasof Protein Sequence and Structure, (1978) vol. 5, suppl. 3, M. O.Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.;Schwartz et al., “Matrices for detecting distant relationships.” inAtlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3, M. O.Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.;Altschul (1991) J. Mol. Biol. 219:555-565; States et al., (1991) Methods3:66-70; Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA89:10915-10919; Altschul et al., (1993) J. Mol. Evol. 36:290-300;ALIGNMENT STATISTICS: Karlin et al., (1990) Proc. Natl. Acad. Sci. USA87:2264-2268; Karlin et al., (1993) Proc. Natl. Acad. Sci. USA90:5873-5877; Dembo et al., (1994) Ann. Prob. 22:2022-2039; andAltschul, S. F. “Evaluating the statistical significance of multipledistinct local alignments.” in Theoretical and Computational Methods inGenome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

The present invention also includes recombinant versions of the solubleform of MDL-1 that bind to MDL-1. Soluble versions can include Ig-MDL-1,Fc-MDL-1, and human serum albumin-MDL-1 fusion proteins. Solublemolecules can also be multimeric forms of MDL-1, e.g., tetramers.Moreover, fragments of the extracellular domain will also providesoluble forms of the MDL-1 protein. Fragments can be prepared usingknown techniques to isolate a desired portion of the extracellularregion.

Conventional molecular biology techniques can be used to producechimeric proteins having MDL-1 fused a heterologous enzymaticallyinactive polypeptide (e.g., a lytic or non-lytic Fc region of IgG, humanserum albumin). Numerous polypeptides are suitable for use asenzymatically inactive proteins in the invention. Preferably, theprotein has a molecular weight of at least 10 kD; a net neutral chargeat pH 6.8; a globular tertiary structure; and of human origin. Where theenzymatically inactive polypeptide is IgG, preferably, the IgG portionis glycosylated. If desired, the enzymatically inactive polypeptide caninclude an IgG hinge region positioned such that the chimeric proteinhas MDL-1 bonded to an IgG hinge region with the hinge region bonded toa longevity-increasing polypeptide. Thus, the hinge region can serve asa spacer between the cytokine and the longevity-increasing polypeptide.A person skilled in molecular biology can readily produce such moleculesfrom an IgG2a-secreting hybridoma (e.g., HB129) or other eukaryoticcells or baculovirus systems. As an alternative to using an IgG hingeregion, a flexible polypeptide spacer, as defined herein, can be used.Using conventional molecular biology techniques, such a polypeptide canbe inserted between MDL-1 and the longevity-increasing polypeptide.

Where the heterologous protein includes an Fc region, the Fc region canbe mutated, if desired, to inhibit its ability to fix complement andbind the Fc receptor with high affinity. For murine IgG Fc, substitutionof Ala residues for Glu 318, Lys 320, and Lys 322 renders the proteinunable to direct ADCC. Substitution of Glu for Leu 235 inhibits theability of the protein to bind the Fc receptor with high affinity.Appropriate mutations for human IgG also are known (see, e.g., Morrisonet al., 1994. The Immunologist 2: 119-124 and Brekke et al., 1994, TheImmunologist 2: 125). Other mutations can also be used to inhibit theseactivities of the protein, and art-recognized methods can be used toassay for the ability of the protein to fix complement or bind the Fcreceptor. Other useful heterologous polypeptides include albumin (e.g.,human serum albumin), transferrin, enzymes such as t-PA which have beeninactivated by mutations, and other proteins with a long circulatinghalf-life and without enzymatic activity in humans.

Preferably, the enzymatically inactive polypeptide used in theproduction of the chimeric protein (e.g., IgG Fc) has, by itself, an invivo circulating half-life greater than that of the extracellularportion of the fusion partner alone (e.g., MDL-1). More preferably, thehalf-life of the chimeric protein is at least 2 times that of thecytokine alone. Most preferably, the half-life of the chimeric proteinis at least 10 times that of the cytokine alone. The circulatinghalf-life of the chimeric protein can be measured in an ELISA of asample of serum obtained from a patient treated with the chimericprotein. In such an ELISA, antibodies directed against the cytokine canbe used as the capture antibodies, and antibodies directed against theenzymatically inactive protein can be used as the detection antibodies,allowing detection of only the chimeric protein in a sample.Conventional methods for performing ELISAs can be used, and a detailedexample of such an ELISA is provided herein.

The chimeric proteins can be synthesized (e.g., in mammalian cells)using conventional methods for protein expression using recombinant DNAtechnology. Because many of the polypeptides used to create the chimericproteins have been previously purified, many of the previously-describedmethods of protein purification should be useful, along with otherconventional methods, for purifying the chimeric proteins of theinvention. If desired, the chimeric protein can be affinity-purifiedaccording to standard protocols with antibodies directed against thecytokine Antibodies directed against the enzymatically inactive proteinare also useful for purifying the chimeric protein by conventionalimmunoaffinity techniques. If desired, the activity of the chimericprotein can be assayed with methods that are commonly used to test theactivity of the protein alone. It is not necessary that the activity ofthe chimeric protein be identical to the activity of the protein alone.

The present invention also includes fusions which include thepolypeptides and polynucleotides of the present invention and a secondpolypeptide or polynucleotide moiety, which may be referred to as a“tag”. The fused polypeptides of the invention may be convenientlyconstructed, for example, by insertion of a polynucleotide of theinvention or fragment thereof into an expression vector as describedabove. The fusions of the invention may include tags which facilitatepurification or detection. Such tags include glutathione-S-transferase(GST), hexahistidine (His6) tags, maltose binding protein (MBP) tags,haemagglutinin (HA) tags, cellulose binding protein (CBP) tags and myctags. Detectable labels or tags such as ³²P, ³⁵S, ¹⁴C, ³H, ^(99m)Tc,¹¹¹In, ⁶⁸Ga, ¹⁸F, ¹²⁵I, ¹³¹I, ^(113m)In, ⁷⁶Br, ⁶⁷Ga, ^(99m)Tc, ¹²³I,¹¹¹In and ⁶⁸Ga may also be used to label the polypeptides of theinvention. Methods for constructing and using such fusions are veryconventional and well known in the art.

Modifications (e.g., post-translational modifications) that occur in apolypeptide often will be a function of how it is made. For polypeptidesmade by expressing a cloned gene in a host, for instance, the nature andextent of the modifications, in large part, will be determined by thehost cell's post-translational modification capacity and themodification signals present in the polypeptide amino acid sequence. Forinstance, as is well known, glycosylation often does not occur inbacterial hosts such as E. coli. Accordingly, when glycosylation isdesired, a polypeptide may be expressed in a glycosylating host,generally a eukaryotic cell. Insect cells often carry outpost-translational glycosylations which are similar to those ofmammalian cells. For this reason, insect cell expression systems havebeen developed to express, efficiently, mammalian proteins having nativepatterns of glycosylation. Alternatively, deglycosylation enzymes may beused to remove carbohydrates attached during production in eukaryoticexpression systems.

Analogs of the MDL-1 peptides of the invention may be prepared bychemical synthesis or by using site-directed mutagenesis, Gillman etal., (1979) Gene 8:81; Roberts et al., (1987) Nature, 328:731 or Innis(Ed.), 1990, PCR Protocols: A Guide to Methods and Applications,Academic Press, New York, N.Y. or the polymerase chain reaction methodPCR; Saiki et al., (1988) Science 239:487, as exemplified by Daughertyet al., (1991) (Nucleic Acids Res. 19:2471) to modify nucleic acidsencoding the peptides. Adding epitope tags for purification or detectionof recombinant products is envisioned.

Protein Purification

Typically, the peptides of the invention may be produced by expressing anucleic acid which encodes the polypeptide in a host cell which is grownin a culture (e.g., liquid culture such as Luria broth). For example,the nucleic acid may be part of a vector (e.g., a plasmid) which ispresent in the host cell. Following expression, the peptides of theinvention may be isolated from the cultured cells. The peptides of thisinvention may be purified by standard methods, including, but notlimited to, salt or alcohol precipitation, affinity chromatography(e.g., used in conjunction with a purification tagged peptide asdiscussed above), preparative disc-gel electrophoresis, isoelectricfocusing, high pressure liquid chromatography (HPLC), reversed-phaseHPLC, gel filtration, cation and anion exchange and partitionchromatography, and countercurrent distribution. Such purificationmethods are very well known in the art and are disclosed, e.g., in“Guide to Protein Purification”, Methods in Enzymology, Vol. 182, M.Deutscher, Ed., 1990, Academic Press, New York, N.Y.

Antibody Structure

In general, the basic antibody structural unit is known to comprise atetramer. Each tetramer includes two identical pairs of polypeptidechains, each pair having one “light” (about 25 kDa) and one “heavy”chain (about 50-70 kDa). The amino-terminal portion of each chain mayinclude a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain may define a constant region primarily responsiblefor effector function. Typically, human light chains are classified askappa and lambda light chains. Furthermore, human heavy chains aretypically classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes).

The variable regions of each light/heavy chain pair may form theantibody binding site. Thus, in general, an intact IgG antibody has twobinding sites. Except in bifunctional or bispecific antibodies, the twobinding sites are, in general, the same.

Normally, the chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are usually alignedby the framework regions, enabling binding to a specific epitope. Ingeneral, from N-terminal to C-terminal, both light and heavy chainscomprise the domains FR1, CDR1, FR2 , CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is, generally, in accordancewith the definitions of Sequences of Proteins of Immunological Interest,Kabat et al.; National Institutes of Health, Bethesda, Md. ; 5^(th) ed.;NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75;Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia et al.,(1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature342:878-883.

Antibody Molecules

The anti-MDL-1 antibody molecules of the invention preferably recognizehuman MDL-1 at the site of Gal9 interaction or antagonize ligandsignaling in another manner. In an embodiment, fully-human monoclonalantibodies directed against MDL-1 are generated using transgenic micecarrying parts of the human immune system rather than the mouse system.These transgenic mice, which may be referred to, herein, as “HuMAb”mice, contain a human immunoglobulin gene miniloci that encodesunrearranged human heavy (μ and γ) and κ light chain immunoglobulinsequences, together with targeted mutations that inactivate theendogenous μ and κ chain loci (Lonberg, N., et al., (1994) Nature368(6474):856-859). These antibodies are also referred to as fully humanantibodies. Accordingly, the mice exhibit reduced expression of mouseIgM or κ, and in response to immunization, the introduced human heavyand light chain transgenes undergo class switching and somatic mutationto generate high affinity human IgGκ monoclonal antibodies (Lonberg, N.,et al., (1994), supra; reviewed in Lonberg, N. (1994) Handbook ofExperimental Pharmacology 113:49-101; Lonberg et al., (1995) Intern.Rev.Immunol. 13:65 -93, and Harding et al., (1995) Ann. N.Y Acad. Sci764:536-546). The preparation of HuMab mice is commonly known in the artand is described, for example, in Taylor et al., (1992) Nucleic AcidsResearch 20:6287-6295; Chen et al., (1993) International Immunology5:647-656; Tuaillon et al., (1993) Proc. Natl. Acad. Sci USA90:3720-3724; Choi et al., (1993) Nature Genetics 4:117-123; Chen etal., (1993) EMBO J. 12:821-830; Tuaillon et al., (1994) J Immunol.152:2912-2920; Lonberg et al., (1994) Nature 368(6474):856-859; Lonberg,N. (1994) Handbook of Experimental Pharmacology 113:49-101; Taylor etal., (1994) International Immunology 6:579-591; Lonberg et al., (1995)Intern. Rev. Immunol. Vol. 13:65-93; Harding et al., (1995) Ann. N.YAcad. Sci 764:536-546; Fishwild et al., (1996) Nature Biotechnology14:845-851 and Harding et al., (1995) Annals NY Acad. Sci. 764:536-546;the contents of all of which are hereby incorporated by reference intheir entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429 and 5,545,807; and International Patent ApplicationPublication Nos. WO 98/24884; WO 94/25585; WO 93/12227; WO 92/22645 andWO 92/03918 the disclosures of all of which are hereby incorporated byreference in their entity.

To generate fully human, monoclonal antibodies to MDL-1, HuMab mice maybe immunized with an antigenic MDL-1 polypeptide as described by Lonberget al., (1994) Nature 368(6474):856-859; Fishwild et al., (1996) NatureBiotechnology 14:845-851 and WO 98/24884. Preferably, the mice will be6-16 weeks of age upon the first immunization. For example, a purifiedpreparation of MDL-1 may be used to immunize the HuMab miceintraperitoneally. The mice may also be immunized with whole cells whichare stably transformed or transfected with an MDL-1 gene.

In general, HuMAb transgenic mice respond well when initially immunizedintraperitoneally (IP) with antigen in complete Freund's adjuvant,followed by every other week IP immunizations (usually, up to a total of6) with antigen in incomplete Freund's adjuvant. Mice may be immunized,first, with cells expressing MDL-1L, then with a soluble fragment ofMDL-land continually receive alternating immunizations with the twoantigens. The immune response may be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma may be screened for the presence of anti-MDL-1antibodies, for example by ELISA, and mice with sufficient titers ofimmunoglobulin may be used for fusions. Mice may be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen. It is expected that 2-3 fusions for each antigen may need to beperformed. Several mice may be immunized for each antigen. For example,a total of twelve HuMAb mice of the HC07 and HC012 strains may beimmunized.

Hybridoma cells which produce the monoclonal anti-MDL-lantibodies may beproduced by methods which are commonly known in the art. These methodsinclude, but are not limited to, the hybridoma technique originallydeveloped by Kohler, et al., (1975) (Nature 256:495-497), as well as thetrioma technique (Hering et al., (1988) Biomed. Biochim. Acta.47:211-216 and Hagiwara et al., (1993) Hum. Antibod. Hybridomas 4:15),the human B-cell hybridoma technique (Kozbor et al., (1983) ImmunologyToday 4:72 and Cote et al., (1983) Proc. Natl. Acad. Sci. U.S.A80:2026-2030), and the EBV-hybridoma technique (Cole et al., inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985). Preferably, mouse splenocytes are isolated and fused with PEG toa mouse myeloma cell line based upon standard protocols. The resultinghybridomas may then be screened for the production of antigen-specificantibodies. For example, single cell suspensions of splenic lymphocytesfrom immunized mice may by fused to one-sixth the number ofP3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50%PEG. Cells may be plated at approximately 2×10⁵ cells/mL in a flatbottom microtiter plate, followed by a two week incubation in selectivemedium containing 20% fetal Clone Serum, 18% “653” conditioned media, 5%origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate,5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and 1× HAT (Sigma; the HAT is added 24hours after the fusion). After two weeks, cells may be cultured inmedium in which the HAT is replaced with HT. Individual wells may thenbe screened by ELISA for human anti-MDL-1 monoclonal IgG antibodies.Once extensive hybridoma growth occurs, medium may be observed usuallyafter 10-14 days. The antibody secreting hybridomas may be replated,screened again, and if still positive for human IgG, anti-MDL-1monoclonal antibodies, may be subcloned at least twice by limitingdilution. The stable subclones may then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

The anti-MDL-lantibody molecules of the present invention may also beproduced recombinantly (e.g., in an E. coli/T7 expression system asdiscussed above). In this embodiment, nucleic acids encoding theantibody molecules of the invention (e.g., V_(H) or V_(L)) may beinserted into a pET-based plasmid and expressed in the E. coli/T7system. There are several methods by which to produce recombinantantibodies which are known in the art. One example of a method forrecombinant production of antibodies is disclosed in U.S. Pat. No.4,816,567 which is herein incorporated by reference. Transformation maybe by any known method for introducing polynucleotides into a host cell.Methods for introduction of heterologous polynucleotides into mammaliancells are well known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene-mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, biolistic injection and directmicroinjection of the DNA into nuclei. In addition, nucleic acidmolecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, forexample, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells,and a number of other cell lines. Mammalian host cells include human,mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells.Cell lines of particular preference are selected through determiningwhich cell lines have high expression levels. Other cell lines that maybe used are insect cell lines, such as Sf9 cells, amphibian cells,bacterial cells, plant cells and fungal cells. When recombinantexpression vectors encoding the heavy chain or antigen-binding fragmentthereof, the light chain and/or antigen-binding fragment thereof areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably, 5secretion of the antibody into the culture medium in which the hostcells are grown.

Antibodies may be recovered from the culture medium using standardprotein purification methods. Further, expression of antibodies of theinvention (or other moieties therefrom) from production cell lines maybe enhanced using a number of known techniques. For example, theglutamine synthetase gene expression system (the GS system) is a commonapproach for enhancing expression under certain conditions. The GSsystem is discussed in whole or part in connection with European PatentNos. 0 216 846, 0 256 055, and 0 323 997 and European Patent ApplicationNo. 89303964.4.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant invention, regardless of the glycosylation of theantibodies.

Antibody fragments, preferably antigen-binding antibody fragments, fallwithin the scope of the present invention also include F(ab)₂ fragmentswhich may be produced by enzymatic cleavage of an IgG by, for example,pepsin. Fab fragments may be produced by, for example, reduction ofF(ab)₂ with dithiothreitol or mercaptoethylamine. A Fab fragment is aV_(L)-C_(L) chain appended to a V_(H)-C_(H1) chain by a disulfidebridge. A F(ab)₂ fragment is two Fab fragments which, in turn, areappended by two disulfide bridges. The Fab portion of an F(ab)₂ moleculeincludes a portion of the F_(c) region between which disulfide bridgesare located.

As is well known, Fv, the minimum antibody fragment which contains acomplete antigen recognition and binding site, consists of a dimer ofone heavy and one light chain variable domain (V_(H)-V_(L)) innon-covalent association. In this configuration that corresponds to theone found in native antibodies the three complementarity determiningregions (CDRs) of each variable domain interact to define an antigenbinding site on the surface of the V_(H)-V_(L) dimer. Collectively, thesix CDRs confer antigen binding specificity to the antibody. Frameworks(FRs) flanking the CDRs have a tertiary structure that is essentiallyconserved in native immunoglobulins of species as diverse as human andmouse. These FRs serve to hold the CDRs in their appropriateorientation. The constant domains are not required for binding function,but may aid in stabilizing V_(H)-V_(L) interaction. Even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughusually at a lower affinity than an entire binding site (Painter,Biochem. 11 (1972), 1327-1337). Hence, said domain of the binding siteof the antibody construct as defined and described in the presentinvention may be a pair of V_(H)-V_(L), V_(H)-V_(H) or V_(L)-V_(L)domains of different immunoglobulins. The order of V_(H) and V_(L)domains within the polypeptide chain is not decisive for the presentinvention, the order of domains given hereinabove may be reversedusually without any loss of function. It is important, however, that theV_(H) and V_(L) domains are arranged so that the antigen binding sitemay properly fold. An F_(v) fragment is a V_(L) or V_(H) region.

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins may be assigned to different classes.There are at least five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2.

The anti-MDL-1 antibody molecules or the MDL-1 soluble proteins of theinvention may also be conjugated to a chemical moiety. The chemicalmoiety may be, inter alia, a polymer, a radionuclide or a cytotoxicfactor. Preferably the chemical moiety is a polymer which increases thehalf-life of the antibody molecule in the body of a subject. Suitablepolymers include, but are not limited to, polyethylene glycol (PEG)(e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol(mPEG). Lee et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEGconjugated single-chain antibodies. Wen et al., (2001) (Bioconj. Chem.12:545-553) disclose conjugating antibodies with PEG which is attachedto a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

The antibodies and antibody fragments or the MDL-1 soluble proteins orfragments thereof of the invention may also be conjugated with labelssuch as ⁹⁹Tc, ⁹⁰Y, ¹¹¹, ³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸F,³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu, ⁶⁷CU, ²¹⁷Ci, ²¹¹At,²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr and ⁵⁶Fe.

The antibodies and antibody fragments, the MDL-1 soluble proteins, MDL-1fusion proteins, or fragments thereof of the invention may also beconjugated with fluorescent or chemilluminescent labels, includingfluorophores such as rare earth chelates, fluorescein and itsderivatives, rhodamine and its derivatives, isothiocyanate,phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label,isoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridimium salt label, an oxalate ester label, an aequorinlabel, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels andstable free radicals.

The antibody molecules or soluble MDL-1 proteins may also be conjugatedto a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosaexotoxin A chain, ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fattyacids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII,and PAP-S, momordica charantia inhibitor, curcin, crotin, saponariaofficinalis inhibitor, mitogellin, restrictocin, phenomycin, andenomycin.

Any method known in the art for conjugating the antibody molecules orprotein molecules of the invention to the various moieties may beemployed, including those methods described by Hunter et al., (1962)Nature 144:945; David et al., (1974) Biochemistry 13:1014; Pain et al.,(1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. andCytochem. 30:407. Methods for conjugating antibodies and proteins areconventional and very well known in the art.

Antigenic (i.e., immunogenic) fragments of the MDL-1 peptides of theinvention are within the scope of the present invention. Antigenicfragments may be joined to other materials, such as fused or covalentlyjoined polypeptides, to be used as immunogens. The antigenic peptidesmay be useful for preparing antibody molecules which recognize MDL-1 orany fragment thereof. An antigen and its fragments may be fused orcovalently linked to a variety of immunogens, such as keyhole limpethemocyanin, bovine serum albumin, or ovalbumin (Coligan et al. (1994)Current Protocols in Immunol., Vol. 2, 9.3-9.4, John Wiley and Sons, NewYork, N.Y.). Peptides of suitable antigenicity may be selected from thepolypeptide target, using an algorithm, see, e.g., Parker et al. (1986)Biochemistry 25:5425-5432; Jameson and Wolf (1988) Cabios 4:181-186;Hopp and Woods (1983) Mol. Immunol. 20:483-489.

Although it is not always necessary, when MDL-1 peptides are used asantigens to elicit antibody production in an immunologically competenthost, smaller antigenic fragments are preferably first rendered moreimmunogenic by cross-linking or concatenation, or by coupling to animmunogenic carrier molecule (i.e., a macromolecule having the propertyof independently eliciting an immunological response in a host animal,such as diptheria toxin or tetanus). Cross-linking or conjugation to acarrier molecule may be required because small polypeptide fragmentssometimes act as haptens (molecules which are capable of specificallybinding to an antibody but incapable of eliciting antibody production,i.e., they are not immunogenic). Conjugation of such fragments to animmunogenic carrier molecule renders them more immunogenic through whatis commonly known as the “carrier effect”.

Carrier molecules include, e.g., proteins and natural or syntheticpolymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides, etc. Protein carrier molecules are especiallypreferred, including, but not limited to, keyhole limpet hemocyanin andmammalian serum proteins such as human or bovine gammaglobulin, human,bovine or rabbit serum albumin, or methylated or other derivatives ofsuch proteins. Other protein carriers will be apparent to those skilledin the art. Preferably, the protein carrier will be foreign to the hostanimal in which antibodies against the fragments are to be elicited.

Covalent coupling to the carrier molecule may be achieved using methodswell known in the art; the exact choice of which will be dictated by thenature of the carrier molecule used. When the immunogenic carriermolecule is a protein, the fragments of the invention may be coupled,e.g., using water-soluble carbodiimides such as dicyclohexylcarbodiimideor glutaraldehyde.

Coupling agents, such as these, may also be used to cross-link thefragments to themselves without the use of a separate carrier molecule.Such cross-linking into aggregates may also increase immunogenicity.Immunogenicity may also be increased by the use of known adjuvants,alone or in combination with coupling or aggregation.

Adjuvants for the vaccination of animals include, but are not limitedto, Adjuvant 65 (containing peanut oil, mannide monooleate and aluminummonostearate); Freund's complete or incomplete adjuvant; mineral gelssuch as aluminum hydroxide, aluminum phosphate and alum; surfactantssuch as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl)propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The polypeptides could also be administered following incorporation intoliposomes or other microcarriers.

Information concerning adjuvants and various aspects of immunoassays aredisclosed, e.g., in the series by P. Tijssen, Practice and Theory ofEnzyme Immunoassays, 3rd Edition, 1987, Elsevier, N.Y. Other usefulreferences covering methods for preparing polyclonal antisera includeMicrobiology, 1969, Hoeber Medical Division, Harper and Row;Landsteiner, Specificity of Serological Reactions, 1962, DoverPublications, New York, and Williams, et al., Methods in Immunology andImmunochemistry, Vol. 1, 1967, Academic Press, New York.

The anti-MDL-1 “antibody molecules” of the invention include, but are byno means not limited to, anti-MDL-1 antibodies (e.g., monoclonalantibodies, polyclonal antibodies, bispecific antibodies andanti-idiotypic antibodies) and fragments, preferably antigen-bindingfragments, thereof, such as Fab antibody fragments, F(ab)₂ antibodyfragments, Fv antibody fragments (e.g., V_(H) or V_(L)), single chain Fvantibody fragments and dsFv antibody fragments. Furthermore, theantibody molecules of the invention may be fully human antibodies, mouseantibodies, rabbit antibodies, chicken antibodies, human/mouse chimericantibodies or humanized antibodies.

The anti-MDL-lantibody molecules of the invention preferably recognizehuman or mouse MDL-1 peptides of the invention; however, the presentinvention includes antibody molecules which recognize MDL-1 peptidesfrom different species, preferably mammals (e.g., non-human primates,pig, rat, rabbit, sheep or dog).

The present invention also includes complexes comprising the MDL=1peptides of the invention and one or more antibody molecules, e.g.,bifunctional antibodies. Such complexes may be made by simply contactingthe antibody molecule with its cognate peptide.

Various methods may be used to make the antibody molecules of theinvention. In preferred embodiments, the antibodies of the invention areproduced by methods which are similar to those disclosed in U.S. Pat.Nos. 5,625,126; 5,877,397; 6,255,458; 6,023,010 and 5,874,299. Hybridomacells which produce monoclonal, fully human anti-MDL-lpeptide antibodiesmay then be produced by methods which are commonly known in the art.These methods include, but are not limited to, the hybridoma techniqueoriginally developed by Kohler et al., (1975) (Nature 256:495-497), aswell as the trioma technique (Hering et al., (1988) Biomed. Biochim.Acta. 47:211-216 and Hagiwara et al., (1993) Hum. Antibod. Hybridomas4:15), the human B-cell hybridoma technique (Kozbor et al., (1983)Immunology Today 4:72 and Cote et al., (1983) Proc. Natl. Acad. Sci.U.S.A. 80:2026-2030), and the EBV-hybridoma technique (Cole et al., inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985). Again, ELISA may be used to determine if hybridoma cells areexpressing anti-MDL-1 peptide antibodies.

Purification of antigen is not necessary for the generation ofantibodies. Immunization may be performed by DNA vector immunization,see, e.g., Wang, et al. (1997) Virology 228:278-284. Alternatively,animals may be immunized with cells bearing the antigen of interest.Splenocytes may then be isolated from the immunized animals, and thesplenocytes may be fused with a myeloma cell line to produce a hybridoma(Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity13:233-242; Preston et al. (1997) Eur. J. Immunol. 27:1911-1918).Resultant hybridomas may be screened for production of the desiredantibody by functional assays or biological assays, that is, assays notdependent on possession of the purified antigen. Immunization with cellsmay prove superior for antibody generation than immunization withpurified antigen (Kaithamana et al. (1999) J. Immunol. 163 :5157 -5164).

Antibody to antigen and ligand to receptor binding properties may bemeasured, e.g., by surface plasmon resonance (Karlsson et al. (1991) J.Immunol. Methods 145:229-240; Neri et al. (1997) Nat. Biotechnol.15:1271-1275; Jonsson et al. (1991) Biotechniques 11:620-627) or bycompetition ELISA (Friguet et al. (1985) J. Immunol. Methods 77:305-319;Hubble (1997) Immunol. Today 18:305-306). Antibodies may be used foraffinity purification to isolate the antibody's target antigen andassociated bound proteins, see, e.g., Wilchek et al. (1984) Meth.Enzymol. 104:3-55.

Antibodies that specifically bind to variants of MDL-1, where thevariant has substantially the same nucleic acid and amino acid sequenceas those recited herein, but possessing substitutions that do notsubstantially affect the functional aspects of the nucleic acid or aminoacid sequence, are within the definition of the contemplated methods.Variants with truncations, deletions, additions, and substitutions ofregions which do not substantially change the biological functions ofthese nucleic acids and polypeptides are within the definition of thecontemplated methods.

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of MDL-1L. Alternatively, bispecificMDL-1 antibodies can bind to another antigen, e.g., DC-SIGN, CD20,RANK-L, etc.

Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy-chain-light-chain pairs,where the two chains have different specificities (Millstein et al.Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al. EMBOJ, 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy-chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light-chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy-chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulinheavy-chain-light-chain pair (providing a second binding specificity) inthe other arm. It was found that this asymmetric structure facilitatesthe separation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690. Forfurther details of generating bispecific antibodies see, for example,Suresh et al. Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al. (1992) J. Exp. Med., 175:217-225 describe theproduction of a fully humanized bispecific antibody F(ab′)₂ molecule.Each Fab′ fragment was separately secreted from E. coli and subjected todirected chemical coupling in vitro to form the bispecific antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe ErbB2 receptor and normal human T cells, as well as trigger thelytic activity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al. (1992) J. Immunol., 148(5):1547-1553.The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al. (1993) Proc. Nall.Acad. Sci. USA, 90:6444-6448 has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al. (1994) J. Immunol., 152:5368.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. (1991) J. Immunol.147: 60.

Uses

The invention provides methods for the screening of compounds thatmodulate the Gal9/MDL-1 interaction and antagonize receptor signaling orfunction. The methods may comprise the screening of a bindingcomposition specific for a polypeptide or nucleic acid of MDL-1, e.g.,an antibody or antigen binding fragment thereof. Control bindingcompositions are also provided, e.g., control antibodies, see, e.g.,Lacey et al. (2003) Arthritis Rheum. 48:103-109; Choy and Panayi (2001)New Engl. J. Med. 344:907-916; Greaves and Weinstein (1995) New Engl. J.Med. 332:581-588; Robert and Kupper (1999) New Engl. J. Med.341:1817-1828; Lebwohl (2003) Lancet 361:1197-1204.

Phosphorylation assays are contemplated to screen for compounds thatmodulate the interaction of Gal9 and MDL-1. For cell basedphosporylation assays, myeloid cells expressing MDL-1 are combined withT cells known to express Tim3 or other proteins that bind Gal9. WhenGal9 and MDL-1 interact, tyrosine phosphorylation of the MDL-1 signalingpartner, DAP12 takes place. A compound of interest is added to the Tcell and myeloid cell mixture. Lysates are prepared and samples areanalyzed for the ability of test compounds to increase or decreasephosphorylation.

Phosphorylation can be analyzed by several means includingimmunoprecipation followed by immunoblotting, ELISA, cell based ELISA,flow cytometry, immunocytochemistry (ICC), immunohistochemistry (IHC;see, e.g., Zell, T. et al. (2001) Proc. Natl. Acad. Sci. USA 98:10805;and Willinger, T. et al. (2005) J. Immunol. 175:5895), and immobilizedmetal affinity chromatography (IMAC; see, e.g., Brill, L. M. et al.(2004) Anal. Chem. 76:2763). Alternatively Gal9 binding to MDL-1 can beassessed using an surface plasmon resonance optical biosensor, such asBiacore (see, e.g., Leonard, et al. (2011) Meth. Mol. Bio. 681:403-418).

The invention also provides a kit comprising a cell and a compartment, akit comprising a cell and a reagent, a kit comprising a cell andinstructions for use or disposal, as well as a kit comprising a cell,compartment, and a reagent.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

EXAMPLES I. General Methods.

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2 d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, N.Y. Methods for proteinpurification include such methods as ammonium sulfate precipitation,column chromatography, electrophoresis, centrifugation, crystallization,and others. See, e.g., Ausubel, et al. (1987 and periodic supplements);Deutscher (1990) “Guide to Protein Purification” in Meth. Enzymol., vol.182, and other volumes in this series; and manufacturer's literature onuse of protein purification products, e.g., Pharmacia, Piscataway, N.J.,or Bio-Rad, Richmond, Calif. Combination with recombinant techniquesallow fusion to appropriate segments, e.g., to a FLAG sequence or anequivalent which can be fused via a protease-removable sequence. See,e.g., Hochuli (1990) “Purification of Recombinant Proteins with MetalChelate Absorbent” in Setlow (ed.) Genetic Engineering, Principle andMethods 12:87-98, Plenum Press, N.Y.; and Crowe, et al. (1992)QIAexpress: The High Level Expression & Protein Purification System,Qiagen, Inc., Chatsworth, Calif.

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the GCG (U. Wisconsin) and GenBanksources. Public sequence databases were also used, e.g., from GenBankand others.

II. Gal9-MDL-1 Pull-down Assay

A pulldown assay employed Invitrogen's M-270 Epoxy Dynabeads bound toMDL-1-Ig or Control-Ig. Beads were incubated for 45 minutes at 4° C.with lysates from fresh bone marrow derived stromal cell line, ST-2.Samples were analyzed by nano-liquid chromatography tandem massspectrometry on the LTQ-Orbitrap to determine unique protein sequences.Galectin-9 was revealed as a key protein which linked specifically withMDL-1Ig fusion protein.

III. Tyrosine Phosphorylation of DAP12

Cell lysates were prepared from MCSF differentiated human macrophagesthat were stimulated with galectin-9, galectin-4, galectin-8, anti-MDL-1(DX246; agonist antibody) or control Ig for 5-15 minutes at 37° C.Lysates were immunoprecipitated with anti-MDL-1 antibody.

Samples were analyzed by western blotting for the presence of tyrosinephosphorylated proteins. Gal9 and DX246 stimulation resulted in DAP12phosphorylation. The blot was stripped and reprobed with anti-Dapl2antibody to confirm the specificity of the phosphorylated protein bands.Samples were also analyzed for the amount of MDL-1 present in eachtreatment sample.

III. Human Osteoclastogenesis Assays

Galectin-9 promotes RankL-independent in vitro differentiation of giantcells and osteoclast-like cells in human peripheral blood CD 14+monocytes cultured in medium containing 30 ng/ml human recombinant M-CSFfor 7 days, in the same manner and with the same kinetics as MDL-1agonistic antibodies.

CD14+ cells were enriched using RosetteSep Human Monocyte EnrichmentCocktail kit and method. Monocytes were cultured in RPMI-10 completemedium containing 30 ng/ml R&D rhM-CSF for 7 days. Cells were diluted to0.4×106 cell/ml in RPMI complete+M-CSF, plated and cultured on 8-wellchambered coverslides for 24 hours. Cells were stimulated the next daywith either 1) recombinant human Gal9 (rhGal9; 10 mg/mL), 2) anti-MDL-1agonist mAb (DX163;10 mg/ml), 3) or an isotype control mAb (msIgGl; 10mg/ml). The cells were stimulated for 2-3 days and then stained foranalysis and photomicrograph recording. Giant cells and osteoclast-likecells were stained in HEMA3 and then with methanoloic Phalloidin-AF594for F-Actin immunofluorescence stain. Images were recorded on a NikonEclipse E600 microscope using a Nuance Fx CRi imaging system.

Both the MDL-1 agonist antibody, DX163, and rhGal9 treatment resulted inthe formation of osteoclast-like cells. Thus, engagement of Gal9 withMDL-1 expressed on myeloid lineage cells induces MDL-1 activity similarto the stimulation by an agonist antibody.

IV. Gal9 Exacerbates Disease in an Murine Antibody Induced Arthritis(AIA) Model

B10.RIII male mice were induced with 3 mg of arthrogen-CIA antibodycocktail (from Chondrex) on day 0. Three groups (n=4) were treated onday 2: 1) naïve, 2) control-Ig (50 μg), or 3) mouse Galectin-9 (fromGalPharma). Arthritic disease progression was monitored for 7 days.

Gal9 treatment resulted in a significant increase of disease progressionover both the naïve and control-Ig treatment groups (see FIG. 1).Further gene expression studies from the treatment groups showed thatGal9 induced upregulation of inflammatory, bone remodeling andtissue-homing chemokine expression.

V. Treatment with MDL-1-Ig Fusion Protein Inhibited IL-23-inducedEnthesopathy

B10.RIII mice were injected with 3 μg of minicircle plasmid DNAcontaining IL-23 gene sequence at day 0. Transgene-induced expression ofIL-23 promoted joint inflammation beginning on day 5. On the day ofdisease onset, mice were treated with 1.0 mg of MDL-1-Ig fusion protein.As seen in FIG. 1, the MDL-1 fusion protein inhibited the interactionbetween MDL-1 positive macrophages and MDL-1L positive cells. Mice weretreated with additional MDL-1-Ig (0.5 mg/dose) at day 10 and 15. n=5mice per group.

IL-23 promotes enthesopathy by activating IL-23R+CD45+ Thyl+ immunecells. This result indicates the MDL-1 may be interact directly orindirectly with CD45+ Thy1+ cells. The data represented by FIG. 1indicates that targeting these cells with an MDL-1 fusion protein willsuppress IL-23-dependent inflammatory disorders.

VI. MDL-1 Tetramers Staining of Lymph Node Cells Identified a Populationof T Lymphocytes that Interact with MDL-1.

PE labeled tetrameric MDL-1 proteins were generated to detect cellsurface interaction with T lymphocytes by flow cytometry. Recombinantmurine MDL-1-Ig fusion protein was manufactured in-house engineered witha BirA (biotin-ligase) targeting site The MDL-1-Ig protein stock wasdesalted using Zeba Spin™ desalting columns 7 kDa MWCO (Pierce)according to the manufacturer's protocol. During the desaltingprocedure, the MDL-1-Ig protein buffer was exchanged for a low-saltbiotinylation buffer (10 mM Tris-HCl, 7.5 mM MgCl₂, 5.0 mM NaCl, pH8.0). Next, 500 μg aliquots of MDL-1-Ig were biotinylated using BirAbiotin-ligase (Avidity) according to the manufacturer's protocol for 1hour at 30° C. The biotinylation reaction was subsequently transferredto a Slide-A-Lyzer™ dialysis cassette, 10 kDa MWCO (Pierce). The proteinwas dialyzed overnight in 2L DPBS at 4° C. to remove excess free biotin.The dialyzed MDL-1-Ig-biotin sample was then concentrated usingVivaspin™500 10 kDa MWCO (GE Healthcare) and quantified using the BCAassay (Thermo). 200 μg of MDL-1-Ig-biotin was aliquoted into a microfugetube for tetramerization. 25 μg of premium grade Streptavidin-PE(Invitrogen) was added to the MDL-1-Ig-biotin sample 10 times. Aftereach addition of Streptavidin-PE, the sample was incubated for 10minutes at room temperature. Following quality analysis by massspectrometry, tetramerized MDL-1-Ig was stored in the dark at 4° C.

Lymph node cells were isolated from naïve BIO.RIII mice. Cells werefirst stained with a vital dye, Fcy receptors (FcγRs) were blocked withFCγR specific antibodies, and the cells were subsequently stained withfluorochrome-labeled anti-CD45 and anti-CD90 mAbs. The resultsrepresented by FIG. 2 showed that about 1% of CD45+, CD90+ lymphoidcells were positive for MDL-1 tetramer staining CD90 negative myeloidcells were negative for MDL-1 tetramer staining

VII. MDL-1-Ig Staining Confirmed CD45, CD90, and CD117 Expression on TLymphocytes that Interact with MDL-1.

Bone marrow was harvested from naïve, wildtype Bl0.RIII mice. Sampleswere first stained with a viability dye, then FcγRs were blocked withantibodies. The Zenon Mouse™ IgG labeling kit (Invitrogen, Cat# Z25152)was used to label MDL-1-Ig reagent with biotin. Bone marrow samples werethen either left unstained, or were stained with 5 mg ofMDL-1-Ig-Biotin. Lastly, all samples were stained with antibodiesagainst CD45, CD90 (Thy1), CD117 (c-kit), as well as withStreptavidin-PE. Bone marrow samples were acquired on the BD LSRII flowcytometer and were analyzed using TreeStar's FlowJo software.

For analysis, bone marrow cells were gated first on size, then onviability to exclude dead cells. The results represented by FIG. 3showed that live cells were further gated on subsets according to CD90and CD45 expression and subsets were then analyzed for backgroundstaining or MDL-1-Ig staining CD117+ CD45+ cells were also gated fromlive cells, and were analyzed for background and MDL1-Ig staining asbefore.

These results indicate that MDL-1L is expressed on a population of Tlymphocytes that also express CD45, CD90, and CD117.

VIII. Depletion of T Lymphocyte Population with MDL-1 Fusion Protein

An MDL-1-Ig fusion protein, specifically, an MDL-1-Ig is used for invitro and in vivo depletetion of the T lymphocyte population expressingCD45, CD90, CD117, and IL-23R. To increase antibody dependent cellularcytoxicity (“ADCC”), the Fc portion of the MDL-1-Ig protein isα-fucosylated as described, e.g., in Sheilds et al. (2002) J. Biol.Chem. 277:26733-26740. Depletion of the T lymphocyte population isanalyzed using standard fluorescent activated cell sorting (FACS)techniques.

What is claimed is:
 1. A method of modulating an interaction between alectin-like molecule expressed on myeloid cells, and a lectin whichbinds to a receptor expressed by a T cell, the method comprising: a)providing a compound that is capable of binding to lectin-like moleculeat a binding site of the lectin; and b) presenting the compound of step(i) to lectin-like molecule and the lectin and thereby modulating theinteraction between the first and second lectin.
 2. The method of claim1, wherein the lectin-like molecule is expressed by a macrophage cell.3. The method of claim 1, wherein the lectin-like molecule is MDL-1 andthe lectin is Gal9.
 4. The method of claim 1, wherein the compound isselected from the group consisting of an antibody, a binding fragment ofan antibody, and a soluble Ig fusion of the lectin-like molecule,wherein the compound modulates the interaction between the lectin-likemolecule and the lectin.
 5. The method of claim 4, where in the compoundbinds MDL-1 and prevents the interaction of the lectin-like moleculewith the lectin.
 6. A method of screening for a compound that modulatesan interaction between a first lectin-like molecule expressed on myeloidcells, and a lectin which binds to a receptor expressed by a T cell, themethod comprising: a) providing a compound that is capable of binding tothe lectin-like molecule at a binding site of the lectin; and b)presenting the compound of step (a) to the lectin-like molecule and thesecond lectin and thereby modulating the interaction between the firstand second lectin.
 7. The method of claim 6, wherein the lectin-like isexpressed by a macrophage cell.
 8. The method of claim 6, whereinlectin-like molecule is MLD-1 and the lectin is Gal9.
 9. The method ofclaim 6, wherein the compound is selected from the group consisting ofan antibody, a binding fragment of an antibody, and a soluble Ig fusionof the lectin-like molecule.
 10. The method of claim 9, wherein theantibody or binding fragment of an antibody inhibits the interaction ofthe lectin-like molecule with the second lectin.
 11. The method of claim10, wherein the antibody or binding fragment of the antibody inhibitsphosphorylation of a signaling molecule associated with the secondlectin.
 12. The method of claim 11, wherein the signaling molecule isselected from the group consisting of DAP12 and Syk and thephosphorylation is tyrosine phosphorylation.
 13. A method of depleting apopulation of T lymphocyte cells comprising contacting the population ofT lymphocyte cells with an MDL-1 fusion protein that binds directly orindirectly to a molecule expressed on the T lymphocyte cells.
 14. Themethod of claim 13, wherein the MDL-1 fusion protein comprises anextracellular domain of MDL-1 and a heterologous protein.
 15. The methodof claim 14, wherein the heterologous protein is an Fc portion of animmunoglobulin molecule.
 16. The method of claim 14, wherein theheterologous protein is human serum albumin.
 17. The method of claim 13,wherein the population of T lymphocyte cells express CD45, CD90, andCD117.
 18. The method of claim 17, wherein the population of Tlymphocyte cells further express IL-23R.
 19. The method of claim 18,wherein the population of T lymphocyte cells mediate enthesopathy.